Synthesis, Characterization And Antitumor Activity Of Copper(II) Complexes, [CuL2] [HL1-3=N,N-Diethyl-N'-(R-Benzoyl)Thiourea (R=H, o-Cl and p-NO2)]

Synthesis, Characterization And Antitumor Activity Of Copper(II) Complexes, [Cul4] [HL=N,N-DiethyI-N’(R-Benzoyl)Thiourea (R=H, o-Cl and p-NO,)] Wilfredo Hermindez a Evgenia Spodine ’ Lothar Beyer b Uwe Schrtider b, Rainer Richter b Jorge Ferreira Mario Pavani a Universidad de Chile, CIMAT, Facultad de Ciencias Quimicas y Farmacuticas, Casilla 233, Santiago 1, Chile b Universitat Leipzig, Institutfiir Anorganische Chemie, Johannisallee 29, D-04103 Leipzig, Germany. Universidad de Chile, Instituto de Ciencias Biomddicas, Programa de Farmacologia Molecular y Clinica, Facultad de Medicina, Casilla 233, ;antiago 1, Chile. GRAPHICAL ABSTRACT The copper(II) complexes of acylthiourea derivatives have been synthesized and characterized by elemental analysis, FT-IR, FAB(+)-MS, NMR(1H, 13C), magnetic measurements and cyclic voltammetry. Assessment of antitumor activity against the mouse mammary adenocarcinoma TA3 cell line has demonstrated that all Cun complexes were more active than their respective ligands and Cu(L3)2 showed a higher cytotoxicity than all the compounds tested. Cellular copper accumulation and DNA binding were also determined and the results are consistent with the cytotoxic activities obtained. 02N’ i// Cu C) 02N Cu(L3)2 Corresponding author. Te1.:+56-2-9782862; fax:+56-2-978-2868; e-mail: 299 Vol. 3, Nos. 3-4, 2005 Synthesis, Characterization And Antitumor Activity of Copper(II) Complexes ABSTRACT The copper (II) complexes (CuL2) were prepared by reaction of Cu(CH3COO)2 with the corresponding derivatives of acylthioureas in a Cu:HL molar ratio of 1:2. Acylthiourea ligands, N,N-diethyl-N’-(R- benzoyl)thiourea (HL j’3) JR=H, o-C1 and p-NO2] were synthesized in high yield (78-83%) and characterized by elemental analysis, infrared spectroscopy, H and 13C NMR spectroscopy. The complexes CuL2 were characterized by elemental analysis, IR, FAB(+)-MS, magnetic susceptibility measurements, EPR and cyclic voltammetry. The crystal structure of the complex Cu(L2)z shows a nearly square-planar geometry with two deprotonated ligands (L) coordinated to Cun through the oxygen and sulfur atoms in a cis arrangement. The antitumor activity of the copper(II) complexes with acylthiourea ligands was evaluated in vitro against the mouse mammary adenocarcinoma TA3 cell line. These complexes exhibited much higher cytotoxic activity (ICs0 values in the range of 3.9-6.9 tM) than their corresponding ligands (40-240 tM), which indicates that the coordination of the chelate ligands around the Cu II enhances the antitumor activity and, furthermore, this result confirmed that the participation of the nitro and chloro substituent groups in the complex activities is slightly relevant. The high accumulation of the complexes Cu(L)2 and Cu(L3)2 in TA3 tumor cells and the much faster binding to cellular DNA than Cu(L1)2 are consistent with the in vitro cytotoxic activities found for these copper complexes. Keywords: Copper(II) complexes; Acylthiourea; Antitumor cytotoxic activity; Cell growth; Cellular DNA. 1. INTRODUCTION Whilst most of the investigations in the treatment of cancer diseases are oriented to synthesize new metal complexes analogous to cis-diamminedichloroplatinum(II) (cisplatin) (antineoplastic agent of clinical use)/1, 2/, there is a growing number of non-platinum metal complexes which also exhibit remarkable anticancer activities/3-5/. Thus, the bis(acetate) bis(imidazole)copper(II) complex has shown a high cytotoxic activity against to the mouse B16 melanoma cancer cell line, and determinations realized with d X174 RF DNA indicate that the target of this complex may be the guanine residues of the DNA helix/6/. In addition, the copper (II) complexes with thiosemicarbazone derivatives (O,N,S-tridentate chelate ligands) are well-known by their biological applications as antiviral/7/, antimicrobial/8/, antitubercular and antitumor agents/9-11/. In this sense, Antholine et al. /12, 13/have reported that the copper complex [{CuL(MeCO2)}.] (HL=2formylpyridine thiosemicarbazone) and related compounds have marked antitumor activities, being more potent than the free ligands against Ehrlich cells injected into mice/14/, Sarcoma 180 ascites tumors/15/and Chinese hamster ovary cells/16, 17/. On the other hand, the biological activities of complexes with thiourea derivatives have been successfully screened for various biological actions/18-21/. Recently, Xu Shen et al. /22/ have reported the antitumor activities of the Mn I complex derived from the desulfurization and hydrolysis of the ligand, N-(p-nitrophenyl)-N’-(methoxycarbonyl) thiourea. This complex showed high 300 W.Hernandez et al. Bioinolganic Chemistry and Applications cytotoxicity in vitro at micromolar concentrations against human ovary tumor 3 AO and mice P388 in vitro. In our previous work, we reported the in vitro antitumor activity of the platinum (II) complexes with analogousligands against mouse mammary adenocarcinoma TA3 cells, where these complexes showed to be more cytotoxic at low micromolar concentrations than their corresponding ligands/23/. Since our earlier work had revealed that platinum complexation with acylthiourea ligands enhances the antitumor activity, we were motivated to use copper as different central atom in order to obtain a cytotoxic behaviour similar to the platinum complexes mentioned before. In the present work, we report about synthesis and characterization of copper(lI) complexes with ligands N,N-diethyl-N’-R-benzoylthiourea [R=H, o-C1 and p-NO2] and their in vitro cytotoxic effect, copper accumulation and binding to cellular DNA on mouse TA3 mammary adenocarcinoma. 2. EXPERIMENTAL 2.1. Materials Copper(II)acetate monohydrate, sodium sulphate, sodium bicarbonate, benzoylchloride, ochlorobenzoylchloride, p-nitrobenzoylchloride and diethylamine from Aldrich were used as received. All solvents were reagent grade and were purified by standard procedures before use. Eagle’s Minimum Essential Medium [MEM(E)] was supplied by Sigma (USA) and the fetal bovine serum [FBS] was provided by Difco (Detroit, MI). The penicillin, streptomycin and sodium chloride 0.9% were obtained from Sanderson’s laboratory (Chile). 2.2. Measurements Elemental analyses were carried out on a Fisons-Carlo Erba 1108 elemental microanalyser. Melting points were determined on a Boetius melting-point apparatus. Magnetic susceptibilities at room temperature (296 K) were measured using the Gouy method on a Johnson-Mathey MSB-MKI balance with HgCo(NCS)4 as standard and diamagnetic corrections were made using Pascal’s constants/24/. The infrared (IR) spectra . (KBr pellets) on a Bruker FT-IR IFS 55 Equinox spectrophotometer in the range FAB(+) mass spectra were obtained on a ZAB-HSQ (V.G. Analytical Ltd.) spectrometer. were recorded in solid state 4000 400 cm Determinations of copper concentration in biological samples were performed on a Perkin Elmer 3110 atomic absorption spectrometer. NMR (H and 3C) spectr (...truncated)